Mn(III) Porphyrin, MnTnBuOE-2-PyP5+, Commonly Known as a Mimic of Superoxide Dismutase Enzyme, Protects Cardiomyocytes from Hypoxia/Reoxygenation Induced Injury via Reducing Oxidative Stress.

Myocardial ischemia-reperfusion injury (I/R) causes damage to cardiomyocytes through oxidative stress and apoptosis. We investigated the cardioprotective effects of MnTnBuOE-2-PyP5+ (BMX-001), a superoxide dismutase mimic, in an in vitro model of I/R injury in H9c2 cardiomyocytes. We found that BMX-001 protected against hypoxia/reoxygenation (H/R)-induced oxidative stress, as evident by a significant reduction in intracellular and mitochondrial superoxide levels. BMX-001 pre-treatment also reduced H/R-induced cardiomyocyte apoptosis, as marked by a reduction in TUNEL-positive cells. We further demonstrated that BMX-001 pre-treatment significantly improved mitochondrial function, particularly O2 consumption, in mouse adult cardiomyocytes subjected to H/R. BMX-001 treatment also attenuated cardiolipin peroxidation, 4-hydroxynonenal (4-HNE) level, and 4-HNE adducted proteins following H/R injury. Finally, the pre-treatment with BMX-001 improved cell viability and lactate dehydrogenase (LDH) activity in H9c2 cells following H/R injury. Our findings suggest that BMX-001 has therapeutic potential as a cardioprotective agent against oxidative stress-induced H/R damage in H9c2 cardiomyocytes.

The Autotaxin-LPA Axis Emerges as a Novel Regulator of Smooth Muscle Cell Phenotypic Modulation during Intimal Hyperplasia.

Neointimal hyperplasia is characterized by a loss of the contractile phenotype of vascular smooth muscle cells (VSMCs). Our group has recently shown that VSMC proliferation and migration are mediated by lysophosphatidic acid (LPA) during restenosis, but the role of autotaxin (ATX; lysophospholipase D), which produces LPA, remains unclear. Endothelial denudation of the mouse carotid artery was performed to induce neointimal hyperplasia, and the extent of damage caused by the ATX-LPA axis was assessed in VSMCs. We observed the upregulation of ATX activity (p < 0.0002) in the injured carotid artery using an AR2 probe fluorescence assay. Further, the tissue carotid LPA levels were elevated 2.7-fold in carotid vessels, augmenting neointimal hyperplasia. We used an electrical cell-substrate impedance sensor (ECIS) to measure VSMC proliferation and migration. Treatment with an ATX inhibitor (PF8380) or LPA receptor inhibitor (Ki16425) attenuated VSMC proliferation (extracellular signal-regulated kinases) activity and migration in response to recombinant ATX. Indeed, PF8380 treatment rescued the aggravated post-wire injury neointima formation of carotid arteries. The upregulation of ATX following vessel injury leads to LPA production in VSMCs, favoring restenosis. Our observations suggest that inhibition of the ATX-LPA axis could be therapeutically targeted in restenosis to minimize VSMC phenotypic modulation and inflammation after vascular injury.

Endothelial Specific Deletion of Autotaxin Improves Stroke Outcomes.

Autotaxin (ATX) is an extracellular secretory enzyme (lysophospholipase D) that catalyzes the hydrolysis of lysophosphatidyl choline to lysophosphatidic acid (LPA). The ATX-LPA axis is a well-known pathological mediator of liver fibrosis, metastasis in cancer, pulmonary fibrosis, atherosclerosis, and neurodegenerative diseases. Additionally, it is believed that LPA may cause vascular permeability. In ischemic stroke, vascular permeability leading to hemorrhagic transformation is a major limitation for therapies and an obstacle to stroke management. Therefore, in this study, we generated an endothelial-specific ATX deletion in mice (ERT2 ATX-/-) to observe stroke outcomes in a mouse stroke model to analyze the role of endothelial ATX. The AR2 probe and Evans Blue staining were used to perform the ATX activity and vascular permeability assays, respectively. Laser speckle imaging was used to observe the cerebral blood flow following stroke. In this study, we observed that stroke outcomes were alleviated with the endothelial deletion of ATX. Permeability and infarct volume were reduced in ERT2 ATX-/- mice compared to ischemia-reperfusion (I/R)-only mice. In addition, the cerebral blood flow was retained in ERT2 ATX-/- compared to I/R mice. The outcomes in the stroke model are alleviated due to the limited LPA concentration, reduced ATX concentration, and ATX activity in ERT2 ATX-/- mice. This study suggests that endothelial-specific ATX leads to increased LPA in the brain vasculature following ischemic-reperfusion and ultimately disrupts vascular permeability, resulting in adverse stroke outcomes.

Alteration of Cellular Energy Metabolism through LPAR2-Axin2 Axis in Gastric Cancer.

Lysophosphatidic acid (LPA), a multifunctional endogenous phospholipid, plays a vital role in cellular homeostasis and the malignant behavior of cancer cells through G-protein-coupled receptors. However, the role of LPA in ß-catenin-mediated gastric cancer is unknown. Here, we have noted the high expression of LPAR2 in human gastric cancer tissues, and that LPA treatment significantly increased the proliferation, migration, and invasion of human gastric cancer cells. Results from our biochemical experiments showed that an LPA exposure increased the expression of ß-catenin and its nuclear localization, increased the phosphorylation of glycogen synthase kinase 3ß (GSK-3ß), decreased the expression of Axin2, and increased the expression of the target genes of the ß-catenin signaling pathway. The LPA2 receptor (LPAR2) antagonist significantly reduced the LPA-induced nuclear localization of ß-catenin, the primary signaling event. The knockdown of LPAR2 in the gastric cancer cell lines robustly reduced the LPA-induced ß-catenin activity. An LPA exposure increased the ATP production by both oxidative phosphorylation and glycolysis, and this effect was abrogated with the addition of an LPAR2 antagonist and XAV393, which stabilizes the Axin and inhibits the ß-catenin signaling pathway. Based on our findings, the possibility that LPA contributes to gastric cancer initiation and progression through the ß-catenin signaling pathway as well as by the dysregulation of the energy metabolism via the LPAR2 receptor and Axin2, respectively, provides a novel insight into the mechanism of and possible therapeutic targets of gastric cancer.

Electrophilic Aldehyde 4-Hydroxy-2-Nonenal Mediated Signaling and Mitochondrial Dysfunction.

Reactive oxygen species (ROS), a by-product of aerobic life, are highly reactive molecules with unpaired electrons. The excess of ROS leads to oxidative stress, instigating the peroxidation of polyunsaturated fatty acids (PUFA) in the lipid membrane through a free radical chain reaction and the formation of the most bioactive aldehyde, known as 4-hydroxynonenal (4-HNE). 4-HNE functions as a signaling molecule and toxic product and acts mainly by forming covalent adducts with nucleophilic functional groups in proteins, nucleic acids, and lipids. The mitochondria have been implicated as a site for 4-HNE generation and adduction. Several studies clarified how 4-HNE affects the mitochondria's functions, including bioenergetics, calcium homeostasis, and mitochondrial dynamics. Our research group has shown that 4-HNE activates mitochondria apoptosis-inducing factor (AIFM2) translocation and facilitates apoptosis in mice and human heart tissue during anti-cancer treatment. Recently, we demonstrated that a deficiency of SOD2 in the conditional-specific cardiac knockout mouse increases ROS, and subsequent production of 4-HNE inside mitochondria leads to the adduction of several mitochondrial respiratory chain complex proteins. Moreover, we highlighted the physiological functions of HNE and discussed their relevance in human pathophysiology and current discoveries concerning 4-HNE effects on mitochondria.

Cryobanking of primary somatic cells of elite farm animals - A pilot study in domesticated water buffalo (Bubalus bubalis).

Buffalo is an important farm animal species in South and South-east Asian countries. Cryopreservation allows long-term storage of somatic cells, which can be made available to research communities. This study aimed to 1) establish and cryopreserve somatic cells from elite buffaloes, and 2) share stored somatic cells and their associated data with researchers. To achieve these targets, somatic cells were established successfully from tail-skin biopsies of 17 buffaloes. The informative data such as buffalo details (breed, date of birth, sex, and age at the time of tissue biopsy collection, and production traits), the number of cryovials stored, and freezing dates were recorded in an electronic file and a printed inventory record. The established somatic cells were flat, spindle-shaped morphology, and expressed vimentin (a fibroblast-like cell type marker) and the negative expression of cytokeratin-18 (an epithelial cell type marker). Altogether, we cryopreserved 970 cryovials (0.1 million cells per vial) from two buffalo breeds, namely Murrah and Nili-Ravi (at least 45 cryovials per animal), for cryobanking. Somatic cell nuclear transfer (SCNT) experiments demonstrated the utility of cryopreserved cells to produce cloned buffaloes. Importantly, these cryopreserved somatic cells are made available to scientific communities. This study encourages the cryopreservation of somatic cells of elite farm animals for their utilization in cell-based research.

Successful cloning of a superior buffalo bull.

Somatic cell nuclear transfer (SCNT) technology provides an opportunity to multiply superior animals that could speed up dissemination of favorable genes into the population. In the present study, we attempted to reproduce a superior breeding bull of Murrah buffalo, the best dairy breed of buffalo, using donor cells that were established from tail-skin biopsy and seminal plasma. We studied several parameters such as cell cycle stages, histone modifications (H3K9ac and H3K27me3) and expression of developmental genes in donor cells to determine their SCNT reprogramming potentials. We successfully produced the cloned bull from an embryo that was produced from the skin-derived cell. Growth, blood hematology, plasma biochemistries, and reproductive organs of the produced cloned bull were found normal. Subsequently, the bull was employed for semen production. Semen parameters such as CASA (Computer Assisted Semen Analysis) variables and in vitro fertilizing ability of sperms of the cloned bull were found similar to non-cloned bulls, including the donor bull. At present, we have 12 live healthy progenies that were produced using artificial insemination of frozen semen of the cloned bull, which indicate that the cloned bull is fertile and can be utilized in the buffalo breeding schemes. Taken together, we demonstrate that SCNT can be used to reproduce superior buffalo bulls.

Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions.

Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12-14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells.

Establishment of a Somatic Cell Bank for Indian Buffalo Breeds and Assessing the Suitability of the Cryopreserved Cells for Somatic Cell Nuclear Transfer.

Biobanks of cryopreserved gametes and embryos of domestic animals have been utilized to spread desired genotypes and to conserve the animal germplasm of endangered breeds. In principle, somatic cells can be used for the same purposes, and for reviving of animals, the somatic cells must be suitable for animal cloning techniques, such as somatic cell nuclear transfer. In the present study, we derived and cryopreserved somatic cells from three breeds of riverine and swamp-like type buffaloes and established a somatic cell bank. In total, 350 cryovials of 14 different individual animals (25 cryovials per animal) were cryopreserved and informative data such as breed value, origin, and others were documented. Immunostaining of the established cells against vimentin and cytokeratin suggested a commitment to the fibroblast lineage. In addition, microsatellite analysis was performed and documented for unambiguous parentage verification of clones in the future. Subsequently, the cryopreserved cells were tested for their suitability as nuclear donors (n = 7) using handmade cloning, and the reconstructed embryos were cultured in vitro. The cleavage rates (95.99% ± 2.17% vs. 82.18% ± 2.50%) and blastocyst rates (37.73% ± 1.54% vs. 24.31% ± 1.78%) were higher (p < 0.05) for riverine buffalo cells than that of swamp-like buffalo cells, whereas the total cell numbers of blastocysts (258.16 ± 36.25 vs. 198.16 ± 36.25, respectively) were similar. In conclusion, we demonstrated the feasibility of biobanking of buffalo somatic cells, and that the cryopreserved cells can be used to produce cloned embryos. This study encourages the development of somatic cell biobanks of domestic livestock, including endangered breeds of buffalo, to preserve valuable genotypes for future revitalization by animal cloning techniques.

Generation of Venus fluorochrome expressing transgenic handmade cloned buffalo embryos using Sleeping Beauty transposon.

The objective of this study was to optimise the electroporation conditions for efficient integration of Venus construct in buffalo fetal fibroblasts using Sleeping Beauty (SB) based transposition and to produce Venus expressing transgenic cloned embryos through handmade cloning (HMC) approach. Primary culture of buffalo fetal fibroblast cells was established and subsequently cultured cells were co-transfected with Venus and helper plasmid at different combinations of electroporation condition. In different combinations of voltage, time and plasmid dose, we observed that 300 V, single pulse for 10 ms in 2 mm cuvette and 1.5-2.0 µg transposons with 200-300 ng transposase dose was optimum for expressing Venus fluorescence in cells via electroporation. After electroporation, the cells were cultured for 2-3 days and then Venus expressing cells were picked with the help of a Pasteur pipette under the fluorescence microscope to enrich them through single cell culture method before using as donor cells for HMC. In vitro matured oocytes were reconstructed with either transfected or non-transfected buffalo somatic cells by electric fusion followed by activation. The reconstructed, activated embryos were cultured in 400 µL of Research Vitro Cleave medium supplemented with 1% fatty acid-free BSA in 4-well dish, covered with mineral oil and incubated in an incubator (5% CO2 in air) at 38.5 °C for 8 days and the developmental competence was observed. The percentage of cleaved, 4-8 and 8-16 cells stage embryos generated through Venus expressing cells were comparable with control, whereas, the morula (21.0 vs 53.0%) and blastocysts (10.5 vs 30.6%) produced through Venus expressing cells was found low as compared to control. These results indicate that fetal fibroblasts transfected with Venus could be used as donor cells for buffalo cloning and that Venus gene can be safely used as a marker of foreign gene in buffalo transgenesis.

Epigenetic status of buffalo fibroblasts treated with sodium butyrate a chromatin remodeling agent.

The somatic cells having higher levels of DNA methylation and reducing it in donor cells before nuclear transfer (NT) by treating them with chemicals, may improve cloning efficiency of NT embryos by providing donor cells with similar epigenetic characteristics as in vivo embryos. Therefore, the present study was planned to understand mechanism of epigenetic changes in donor cells (buffalo fibroblasts) treated with different concentration of sodium butyrate (NaBu)-a chromatin remodeling agent. The cultured fibroblasts purity and lineage were confirmed by fibroblast specific protein and gene markers (Vimentin, Tubulin and Cytokeratin) at different passages using immuno-staining and qPCR respectively. The buffalo fibroblast cells were treated with 1, 3 and 5 mM of NaBu and observations were taken on their morphological changes, population doubling time and cell proliferation after 48 h of treatment. The epigenetic changes were observed using acetylation (H3K9ac) and methylation (H3K27me3) markers expression. The fibroblast cells derived from new born ear tissue started emerging and anchoring to cell culture flasks within 24 h and showed spindle-shaped morphology. The confluent culture was cryopreserved at different time interval. The post thaw culture behavior of the cryopreserved cells was also observed at different time interval and passages. The morphology of NaBu treated cells were changed with increase of dosages of NaBu treatment. The increase population doubling times and decreases the proliferation rate in the dose dependent manner. The NaBu treatment showed that the significantly increased the acetylation (H3K9ac) and methylation (H3K27me3) level over the control. Based on the observations of fibroblast characterization as well as epigenetic modifications of these cells after treatment with NaBu, results suggest that the cells may provide a useful approach for better epigenetic reprogramming in SCNT embryos.